Enterococci are one of the leading causes of nosocomial infections and cause a variety of disease states including endocarditis, bacteremia, meningitis, wound infections, and urinary tract infections. Enterococcal and other Gram positive infections are becoming increasingly difficult to treat due to a rising prevalence of resistance to antibiotics of last resort. E. faecalis also serves as a vector for the spread of antibiotic resistance determinants through conjugal transfer to other bacterial species. Thus, it is of great interest to understand fundamental biological processes of this organism with the aim of identifying novel therapeutic targets. It is becoming increasingly evident that focally localized assembly of surface-exposed proteins is a general strategy among Gram positive pathogens and is strongly associated with microbial virulence. However, little is known about how the focal surface protein assembly machinery itself localizes. Therefore, the objective of this project is to determine the mechanisms underlying localized virulence factor assembly and assess whether these sites can be specifically targeted by antimicrobials. My research aims to clarify the conceptual details and implications of localized virulence factor assembly for Gram positive infection and therapy using two complementary approaches. First, I will determine the molecular components that guide focal localization and retention at single domains using classical and chemical genetic methods to manipulate localization patterns of secretion and sorting proteins, coupled with biochemical and microscopic assays for focal interactions. Second, I will assess the functional consequence of perturbing focal localization for virulence factor assembly read out at the single cell level, the bacterial population level, and in models of disease. The virulence factor and model organism of choice for these studies is the sortase-assembled pilus of E. faecalis because of their outstanding clinical importance. A more thorough understanding of focally localized virulence factor assembly will be achieved in 3 aims designed to: 1) define structural components of the localized complex, 2) elucidate subcellular assembly mechanisms, and 3) characterize the role of localization in virulence factor function and virulence. The accessibility of localized sites of Enterococcal secretion and sorting to the extracellular environment and its critical role in processing and secreting virulence factors make it a likely site for both localized interaction between the bacterium and the host (thus supporting virulence) and for targeting by antivirulence and antimicrobial agents (thus representing a vulnerable target). Therefore, this research has implications for both fundamental bacterial processes and also for host-pathogen interactions and therapy of an important disease agent. I obtained a Masters in Public Health and a PhD at Northwestern University, training with Dr. Hank Seifert on the contribution of pili to disease caused by the Gram-negative bacterium Neisseria gonorrhoeae. I chose to couple these two degrees because I wanted to approach the problem of gonorrhea from a multi-dimensional standpoint of understanding bacterial strategies for pathogenesis as well as implications of those particular strategies for human disease. I capitalized on my expertise in molecular and epidemiological perspectives on infectious disease with existing expertise in the Hultgren lab in the structural basis of pilus assembly and function, mammalian models of pathogenesis, and post-genomic analysis of bacterial pathogens. Working at Washington University has also enabled me to draw on the mentorship and experience of multiple scientists, including the previous chair of the department, Staffan Normark, an expert on Gram positive pilus assembly, protein secretion, and cell wall biogenesis. I have developed fruitful collaborative and mentorship relationships with current faculty members as well, including Dr. Amanda Lewis who has provided scientific expertise as a Gram positive microbiologist and professional guidance as the newest member of our faculty. I have been able to develop a new line of research within the Hultgren lab that synthesizes my experience with all of my mentors and aims to elucidate the general role of coordinated protein secretion and trafficking in both basic Gram positive physiology as well as Gram positive infections of humans. I continue to deliberately focus on fundamental concepts as they relate to disease. I plan to utilize the remaining time of my mentored training to become expert in biochemical techniques important for the continued expansion of my research interests. During my postdoctoral period, I have received formal and informal training in writing and oral presentation, lab management and mentoring skills, and in the ethical conduct of research. I will continue to develop these skills during the remainder of my postdoctoral training. After acquiring this expertise, I will transition into an independent faculty position, pursuing this research program full time at a major research university. A K99/R00 award is thus ideally suited for my current situation and would be a decided advantage in both providing me additional mentored time to acquire specific technical training and facilitating my transition into an independent investigator. PUBLIC HEALTH RELEVANCE STATEMENT: Enterococcus faecalis is a bacterium that causes infections in sites ranging from the heart to the blood to the urinary tract, most often in already ill and hospitalized patients. This project will investigate the way these bacteria builds "tools" to attach to human tissues during infection. The goal of these studies is to find new antimicrobial agents that can target these bacterial "tools" and help prevent infection in humans.